Amis Pacilo and Yami Cipoho are not the same as the Pacific breadfruit starch crop—Target enrichment phylogenomics of a long-misidentified Artocarpus species sheds light on the northward Austronesian migration from the Philippines to Taiwan

‘Breadfruit’ is a common tree species in Taiwan. In the indigenous Austronesian Amis culture of eastern Taiwan, ‘breadfruit’ is known as Pacilo, and its fruits are consumed as food. On Lanyu (Botel Tobago) where the indigenous Yami people live, ‘breadfruit’ is called Cipoho and used for constructing houses and plank-boats. Elsewhere in Taiwan, ‘breadfruit’ is also a common ornamental tree. As an essential component of traditional Yami culture, Cipoho has long been assumed to have been transported from the Batanes Island of the Philippines to Lanyu. As such, it represents a commensal species that potentially can be used to test the hypothesis of the northward Austronesian migration ‘into’ Taiwan. However, recent phylogenomic studies using target enrichment show that Taiwanese ‘breadfruit’ might not be the same as the Pacific breadfruit (Artocarpus altilis), which was domesticated in Oceania and widely cultivated throughout the tropics. To resolve persistent misidentification of this culturally and economically important tree species of Taiwan, we sampled 36 trees of Taiwanese Artocarpus and used the Moraceae probe set to enrich 529 nuclear genes. Along with 28 archived Artocarpus sequence datasets (representing a dozen taxa from all subgenera), phylogenomic analyses showed that all Taiwanese ‘breadfruit’ samples, together with a cultivated ornamental tree from Hawaii, form a fully supported clade within the A. treculianus complex, which is composed only of endemic Philippine species. Morphologically, the Taiwanese ‘breadfruit’ matches the characters of A. treculianus. Within the Taiwanese samples of A. treculianus, Amis samples form a fully supported clade derived from within the paraphyletic grade composed of Yami samples, suggesting a Lanyu origin. Results of our target enrichment phylogenomics are consistent with the scenario that Cipoho was transported northward from the Philippines to Lanyu by Yami ancestors, though the possibility that A. treculianus is native to Lanyu cannot be ruled out completely.

Introduction specimens, favored a broad approach that encompasses breadfruit's wild relatives. However, Zerega et al. 2005 [38], who worked with fresh specimens, molecular data, as well as herbarium specimens, took a narrower approach, recognizing A. camansi Blanco (breadnut) and A. mariannensis Trécul (dugdug) as distinct, closely related species of the cultivated breadfruit A. altilis. This latter approach has been widely adopted [32,34,45], including the current study.
In Taiwan, the local name for 'breadfruit' is "麵包樹 miao-bao-shu" (where "mian-bao" means bread and "shu" means tree in Mandarin Chinese), which is a common tree species occurring both in cultivation throughout much of the island and in forests of Lanyu [46]. The earliest traceable record of 'breadfruit' of Taiwan appeared in the diary (6 th September 1890) of Rev. George Leslie Mackay  [47], a Canadian Presbyterian missionary who had profound influence on Taiwan's Christianity and medical education [48]. Mackay recorded "Pat-chi-lút Bread fruit tree" from Chhit-kha-chhoan [47] (Fig 1), a Lam-si-hoan (Amis) village in eastern Taiwan [49]. In his memoir "From Far Formosa", Mackay wrote that "Breadfruit (Artocarpus incisa)" was "used by the aborigines exclusively" [50]. In Amis' oral history, Pacilo (�Pat-chi-lút) [51] (also known as 'Apalo [51] / Qapalo [14], or Facidol called by central and southern Amis [14,30]) was said to be introduced by Amis ancestors from overseas [52]. Nowadays, Pacilo is still commonly planted in eastern Taiwan especially around Amis households as food [51] and has become a popular ethnic cuisine of eastern Taiwan.
In Yami language, 'breadfruit' is called Cipoho or Ciporo, almost identical to Cipoho or Tipoho of the other Batanic languages [14]. In forests managed by Yami people, Cipoho is the third most dominant tree species [53]; traditionally, however, fruits of Cipoho are rarely consumed as food on Lanyu [54,55]. Instead, Yami people use the light red wood of Cipoho for making houses, hats, plates, mortars (for crushing millet seeds), and the plank-boats Tatala and Cinedkeran that are central to the Yami culture [55][56][57][58][59]. Given the phonetic similarity of the names for 'breadfruit' in Batanic languages [14] and its appearance as a timber tree in managed and semi-naturalized forests of Lanyu [53, 60, 61], Cipoho has long been regarded as non-native [30,61], possibly introduced by Yami ancestors along with their northward migration from Batanes Island [30].
However, in the earliest record of Lanyu's 'breadfruit', which was documented by Yasusada Tashiro [60] in 1900 A.D. during the early period of the Japanese occupation, the species appeared to have been confused with the jackfruit "Artocarpus integrifolia L." (= A. heterophyllus Lam.), which was introduced during the Dutch colonization (1624-1662 A.D.) and has been widely grown in Taiwan since [52,62]. In 1900 A.D., Tashiro was amongst the earliest botanists who ever visited Botel Tobago, and in "A guide to planting trees in urban Taiwan," he recorded 'breadfruit' as growing naturally and abundantly on Botel Tobago and commonly planted around indigenous villages of eastern Taiwan [60]. To promote 'breadfruit' as an urban tree species suitable even in northern Taiwan, Tashiro also referred to a 'breadfruit' tree grown in Mackay's residence in Tamsui (Fig 1) where he lived during his mission. Based on a widely circulated anecdote, that vibrant tree was grown from a Pat-chi-lút seedling given to Mackay by the Nan-shih Amis [63]. Likely due to Tashiro's promotion [60,64], 'breadfruit' is now widely cultivated throughout Taiwan and is amongst the most common tree species planted on school campuses [65][66][67]. Because 'breadfruit' trees are easily accessible, its natural compounds and pharmaceutical properties have been extensively studied in Taiwan [68][69][70].
[85]. These samples were within the 'Philippinensi' clade of Sect. Artocarpus, Subgenus Artocarpus, a clade comprising species thought to be endemic to the Philippines [86]. Since A. treculianus (including A. ovatifolius Merr.) has long been considered endemic to the Philippines [43, 45, 86], the likely persistent misidentification of the Taiwanese 'breadfruit' prompted us to conduct the present study. Additionally, considering the close geographic proximity and phytogeographic similarity between Lanyu and the Philippines [87] (Fig 1), the native status of A. treculianus has to be reconsidered. Indeed, in the earliest record by Tashiro [60], Cipoho was regarded as a native species on Lanyu, though latter scholars assumed that the species was not native since it was the "breadfruit" [30,61]. By taking advantage of recent phylogenomic studies in Artocarpus [88] using Hyb-Seq [85, 86, 89], we asked the following questions: (1) How many species of Artocarpus constitute the 'breadfruit' species of Taiwan and what is the correct name of the most common 'breadfruit' of Taiwan? (2) Is Cipoho native to Lanyu? and (3) What are the likely geographic origins of these 'breadfruit' species? By answering these questions, we also hope to contribute to our understanding of Yami's ancestry.

Sampling
In Taiwan, the "breadfruit" is considered to be Artocarpus incisus (Thunb.) L.f. (= A. altilis), which in "The Red List of Vascular Plants of Taiwan 2017" is listed as "Least Concern" [84]. So only oral permissions from local land owners were needed to sample the species. Since there is a strong indigenous consciousness on Lanyu, we also obtained a collecting permit from the Lanyu Township Office. Prior to sampling, we thoroughly examined specimens in major herbaria (TAI, TAIF, TNM, and HAST [90]), literature [46,72,74,76,79,81], and online information pertaining to the 'breadfruit' of Taiwan. Our preliminary investigation concluded that the morphology of almost all Taiwanese 'breadfruit' trees matched A. treculianus as recently circumscribed by Gardner and Zerega 2021 [86], rather than the true Pacific breadfruit, A. altilis. Because A. treculianus is considered a species complex and its taxonomy remains unsettled [86], we proceeded to sample 'breadfruit' from multiple localities of Taiwan, with a special focus on the traditional territory of the Amis of eastern Taiwan and Lanyu (Fig 1). In addition to the Taiwanese 'breadfruit', a genuine A. altilis, which is extremely rare in Taiwan, was located in National Pingtung University (NPTU) and sampled (Fig 2). We also sampled one jackfruit (A. heterophyllus), one A. odoratissimus Blanco [91], and one A. xanthocarpus from National Chiayi University (NCYU), National Pingtung University of Science and Technology (NPUST), and National Museum of Natural Science (NMNS), respectively, to improve Artocarpus sampling within Taiwan. Voucher information of the 36 samples collected in current study is detailed in Table 1. Voucher specimens were deposited in the Herbarium of Academia Sinica, Taiwan (HAST). To assure species identification of our samples, 28 already-sequenced accessions (S1 Table)

Sequence assembly and phylogenetic analysis
The sequencing quality of raw reads of 36 newly sequenced and 28 archived samples were evaluated using FastQC v0.11. 5

Results and discussion
A total of 30,843 Mb of raw reads were acquired for the 36 newly sequenced samples, with an average of 857 Mb per sample. For the 28 archived samples, a total of ca. 6,804 Mb of raw reads

PLOS ONE
The 'breadfruit' of Taiwan is not Artocarpus altilis but A. treculianus were downloaded, with an average of ca. 243 Mb per sample. Statistics of the assembled quality and summaries of the exon and supercontig alignments are detailed in S1 In ASTRAL trees reconstructed based on both exons and the supercontigs (Fig 3), phylogenetic relationships of all major clades are fully supported and identical to those proposed in Gardner (Fig 3) in Taiwan. Specifically, within Sect. Duricarpus/Clade 'Asperifolii', AoMYT20 and NZ618 (A. odoratissimus s.s. "Lumok") form a clade sister to the clade of A. mutabilis "Pingan", indicating that the Taiwanese A. odoratissimus s.l. is the Lumok [91].
Taxonomy of the A. treculianus complex remains unsettled [86], as there is considerable variation in leaf shape and pubescence, as well as the length of staminate inflorescence (ranging from 1-21 cm), and in the shape of the syncarp. Nevertheless, morphology of the Taiwanese 'breadfruit' sampled in the current study (Fig 4) differs from the closely allied A. blancoi and matches A. treculianus as circumscribed by Gardner and Zerega 2021 [86], confirming our preliminary identification. It is important to note that our current understanding of A. treculianus has been based on limited herbarium collections, which often do not well represent the morphological variation, even within an individual tree. For instance, leaves of A. treculianus observed in Taiwan often show considerable variation during different developmental stages (Fig 4L) as well as within a single tree (Fig 4M). An even greater variation is observed in staminate inflorescences (Fig 5), which can be important in the classification of Artocarpus species [86].
Since Jarrett's [43] seminal taxonomic work, Artocarpus treculianus has been regarded as an endemic taxon of the Philippines, with its northern distributional limit in the Batanes Islands [45,86] where this "vulnerable" species grows both around human habitations as well  Table 1

PLOS ONE
The 'breadfruit' of Taiwan is not Artocarpus altilis but A. treculianus

PLOS ONE
The 'breadfruit' of Taiwan is not Artocarpus altilis but A. treculianus as "in secondary forests and edges of forests" [105]. As a popular shade tree, its fruits are also eaten and leaves of this tree (Fig 4K) are also eaten and used as plates and to wrap food during festivities [105]. Additionally, its wood is used for making Bangka (a type of fishing boat common in the Philippine coastal villages), Bangka trusses, and doors and windows of traditional houses. Moreover, its white latex is cooked and used as bird-trap [105]. All these traditional utilities are highly similar to the traditional Yami culture [29,55,59,75]. The presence of A. treculianus in Lanyu thus is consistent with the scenario of a northward transport by ancestors of speakers of the Batanic languages, providing a commensal species likely brought by the 'into Taiwan' Austronesian migration [14,30]. On the other hand, the possibility that A. treculianus is a native species of Lanyu cannot be ruled out, given the close geographical proximity and phytogeographic similarity between Lanyu and the Philippines

PLOS ONE
The 'breadfruit' of Taiwan is not Artocarpus altilis but A. treculianus 185 km-wide Bashi Channel (Fig 1) separating Lanyu and the Batanes Island is highly plausible. To test these alternative scenarios, further sampling in the Batanes Islands and from throughout the Philippines is essential.
Despite limited sampling, our ASTRAL trees reveal a marked phylogeographic structure. Considering just the samples collected from traditional territories of Amis and Yami (boldfaced in Fig 3), the Amis samples form a fully supported clade (i.e., Pacilo clade) deriving from a paraphyletic grade consisting of the Lanyu samples (i.e., Cipoho grade), suggesting that the Amis samples originated from Lanyu. This inference is consistent with the Amis tale that their ancestors came from the overseas islands Sanasai (i.e., Lyudao, an island. 73 km north of Lanyu; Fig 1) and/or Vutul (i.e., Botel Tobago) [107, 108]. On the contrary, the lack of structure within the Pacilo clade suggests that this popular fruit crop of the Amis people may have been extensively translocated across eastern Taiwan. Additionally, within the Pacilo clade, the generally poor support values and highly discordant and entangled relationships between the exon and supercontig trees indicate the reticulated nature of tokogenetic relationships. Nevertheless, based on the ASTRAL trees (Fig 3), it is evident that A. treculianus cultivated in CYBG (CCR208 and 209), NMNS (CCR205), and NTU (CCR179) originated from Lanyu, while trees planted in AS (CCR089), TPBG (CCR182 and 184), NPTU (CCR202), and other localities (CCR195,198,and 203,and CHW72 and 75) were derived from eastern Taiwan (Fig 1), indicating multiple introductions of the 'breadfruit' to various parts of the island. Additionally, the placement of NZ203, a sample cultivated at the Fort de Russey Park in Honolulu, suggest a possible Lanyu origin. Our data thus demonstrate that the Artocarpus-Moraceae probe set [89] is efficient in detecting phylogeographic structure even on a small geographic scale, providing a genomic tool for unraveling the origin of A. treculianus with a more comprehensive sampling.
The correction of a persistent misidentification of the Taiwanese 'breadfruit' also underscores the possibility that the taxonomy of many common species might still be problematic. Unlike rare species, common plants may be less attractive for taxonomic research because their taxonomy is usually just assumed to be well-studied. On the other hand, because common species are often easily accessible, they are often used for applied research. For instance, a quick 'Google Scholar' search with the key words 'breadfruit' and 'Taiwan' resulted in more than 30 research articles of natural compound and pharmaceutical studies of either 'Artocarpus altilis' or 'A. communis', which were all mis-identifications of A. treculianus (S1 Appendix). This has major impacts on downstream use of such data! Although A. treculianus and A. altilis are both members of Sect. Artocarpus, the crown age of the diversification of the two species was estimated at ca. 25

Conclusions
Our phylogenomic analysis (Fig 3) based on 529 nuclear genes enriched using the Artocarpus-Moraceae probes [89] confirms that the correct name for the Taiwanese 'breadfruit' is Artocarpus treculianus Elmer (Fig 4) according to the most updated taxonomic revision [86], while the true breadfruit, A. altilis, is extremely rare in Taiwan (Fig 2). Since A. treculianus is thought to be endemic to the Philippines, our study suggests that the species was likely transported from the Philippines to Lanyu in prehistorical time, providing a candidate study system to test the northward Austronesian migration into Taiwan using a commensal approach. Our study and Chang-Fu Hsieh, Szuwei Tsai, Gene-Sheng Tung, Chih-Kai Yang, and Alex Hon-Tsen Yu for valuable suggestions and discussions.